percoll使用方法

Product Information PERCOLLProduct Number P1644, P4937, and P7828Storage Temperature 2-8°CCas #: 65455-52-9Product DescriptionAppearance: Clear colorless to very faint yellow liquid Refractive index: 1.3540 (+ or -) 0.005 at 20°C Viscosity: 10 (+ or -) 5 centipoise at 20°C Osmolality: < 25 mOs/kg H2OConductivity: < 1.0 mS/cmDensity: 1.130 +/- 0.005 g/mlPercoll is a well referenced medium for density gradient centrifugation of cells, viruses and subcellular particles. Percoll consists of colloidal silica particles of 15-30 nm diameter (23% w/w in water) which have been coatedwith polyvinylpyrrolidone (PVP). The PVP coatingrenders the product completely non-toxic and ideal foruse with biological materials. The PVP is firmly boundto the silica particles as a monomolecular layer. Due toits heterogeneity in particle size, sedimentation occursat different rates, spontaneously creating very smooth, isometric gradients in the range of 1.0-1.3 g/ml. Most biological particles having sedimentation coefficientvalues greater than 60S can be successfully isolated in Percoll gradients.Preparation InstructionsPercoll is best used in balance salt solutions,physiological saline or 0.25 M sucrose. Cells can be separated in gradients in balanced salts solutions. Subcellular particles, however, tend to aggregate in the presence of salts and it is recommended that the separation of such particles be carried out in Percolldiluted with sucrose (0.25 M final concentration).The low osmolality of Percoll permits this parameter tobe controlled by the user without interference from the density medium itself. The addition of 9 parts (v/v) of Percoll to one part (v/v) of either 1.5 M NaCl, 10 X concentrated culture medium or 2.5 M sucrose willresult in a solution adjusted to approximately 340mOsm/kg H2O. Final adjustments can be made withthe addition of salts or distilled water. The precise osmolality should be checked prior to use with an osmometer.Percoll can be used within the pH range of 5.5 to 10.0 without any changes in properties. Percoll may form agel at pH values below 5.5. Gelling can also be causedby the presence of divalent cations, particularly atelevated temperatures. Percoll will form self-generated gradients bycentrifugation at 10,000g in 0.15 M saline or 25,000 gin0.25 M sucrose in fixed angle rotors after 15 minutes.Cells or subcellular particles can be mixed with Percollprior to centrifugation and will band isopycnically as the gradient is formed in situ. Although Percoll is best usedin angle-head rotors, banding of cells on preformedgradients can be carried out at 400 g for 20 to 30minutes in swing-out rotors.Percoll may be diluted directly to make a final workingsolution of known density by the following procedure.In a graduated cylinder, add 1.5 M NaCl or 2.5 Msucrose to 1/10 the desired volume. To this add therequired volume of undiluted Percoll, calculated usingthe formula below. Make up to the final volume withdistilled water.V o = V (d – 0.1 d10 – 0.9 )d o – 1V o = Volume of Percoll required (undiluted Percoll) in mlV = Volume of final working solution in mld = Desired density of final working solutiond o = Density of Percoll undilutedd10= Density of 1.5 M NaCl (1.058) or 2.5 M sucrose(1.316)The above formula is useful for achieving densities thatwill be very close to the actual densities required.However, slight variations in densities and volumesmay affect final density. For highly accurate density requirements, it is recommended to check and adjustthe final density using a densitometer or refractometer.Sigma offers a Percoll density marker bead kit (Sigmaproduct # DMB-10) which contains ten color-codeddensity markers covering the density range of 1.017-1.142 in saline or 1.037-1.136 in sucrose. The densityof each marker is calibrated to +/- 0.0005 g/ml. Storage/StabilityPercoll is supplied sterile and can be stored for up totwo years in an unopened container. At –20°C, it canonly be stored approximately six months. If stored at–20°C, gradients form upon thawing, necessitatingmixing the contents of the bottle before use. Preformed3050 Spruce StreetSaint Louis, Missouri 63103 USATelephone 800-325-5832 • (314) 771-5765Fax 800-325-5052 • (314) 286-3828email: sigma-techserv@Internet: gradients can be stored for weeks without a change in gradient shape, provided that the gradient is sterile and not physically disturbed. It may be autoclaved at120C for 30 minutes without any change in properties. Autoclaving of Percoll solutions must be carried out without addition of salts or sucrose. The presence of salts will cause Percoll to gelatinize and the presence of sucrose will cause caramelization. Minimum contact with air should be maintained during autoclaving to avoid formation of solid particles at the Percoll/air interface. This can be accomplished by using a narrow-top bottle. If particles do form, they may be removed by filtration or low speed centrifugation. If any significant evaporation occurs during autoclaving, the volume should be replenished with sterile water so that the density is not affected. Percoll cannot be sterile filtered. The plastic bottles in which Percoll is packaged are not autoclavable..ProcedureExamples of Separations in PercollCentrifugationSource Density (g/ml) ConditionsRat Liver CellsHepatocytes 1.07-1.10 30,000g-30minKupffer cells 1.05-1.06 30,000g-30minHuman CellsThrombocytes 1.04-1.06 *Lymphocytes 1.06-1.08 *Granulocytes 1.08-1.09 *Erythrocytes 1.09-1.10 *E. coli 1.13 30,000g-20 minVirusTobacco mosaic 1.06 100,000g-45minEquine abortion 1.08 40,000g-45minInfluenza 1.06 25,000g-25min OrganellesMitochondria 1.09-1.11 50,000g-45min Lysozomes 1.04-1.07 50,000g-45min1,08-1.11 50,000g-45min Peroxisomes 1.05-1.07 63,000-30min Synaptosomes 1.04-1.06 50,000g-45minNuclei 1.08-1.12 100,000g-60min* Separation of blood cells is best carried out by pre-forming the gradient (starting density 1.09 g/ml) by centrifugation at 20,000g for 20 minutes, then layering Blood on top of the gradient. Then centrifuge at 1,000g for 5 minutes in a swinging-bucket rotor, leaving the thrombocytes in the serum layer above the gradient; the serum layer can be removed with a pipette (rate-zonal separation). A further spin for 20 minutes at 1,000 g separates the other cell types at their isopycnic densities.After centrifugation, Percoll can be fractionated by puncturing a hole in the bottom of the tube. A simple and convenient method is to collect the fractions from the tube by displacement with a dense medium such as undiluted Percoll or a 60-65% solution of sucrose.Percoll does not interfere with fluorescent activated cell sorting, or with electronic cell counting instruments.Removal of Percoll from cellsLiving cells can be separated from Percoll medium by washing with physiological saline (5 volumes to1 volume of cell suspension). The washing may be repeated two to three times and the cells collected between each washing step by centrifugation at 200 g for 2-10 minutes.For viruses and subcellular particles which are too small to be pelleted by low speed centrifugation, the particles can be separated from Percoll by high-speed centrifugation. The undiluted fraction is centrifuged at 100,000 g for two hours in a swinging-bucket rotor or 90 minutes in an angle-head rotor. The biological material remains above the hard pellet of Percoll. References* Percoll is a product of Pharmacia.Information on physical properties and applications was obtained from our supplier.Lymphocyte SeparationStibenz, D. and Buhrer, C., Scand. J. Immunol., 39, 59-63, 1994.Pistoia, V. et al, Stem Cells, 11, 150-155 (1993). Giddings, J.C. et al., Clin. Lab. Hematol., 2, 121-128 (1980).MonocytesPrzepiorka, D et al., Am. J. Clin. Pathol., 95, 201-206 (1991).Osipovich, O.A. et al., In vitro. Biull. Eskp. Biol. Med, 113, 638-640 (1992).Giddings, J.C. et al., Clin. Lab. Hematol., 2, 121-128(1980).ErythrocytesPascual, M. et al., Eur. J. Immunol., 24, 702-708 (1994).Vanden Berg, J.J.M. et al., Arch. Biochem. Biophys., 298, 651-657 (1992).Rennie, C. et al., Clin. Chim. Acta, 98, 119-125 (1979). Natural Killer CellsWarren, H.S. and Skipsey, L.J., Immunol., 74, 78-85 (1991).Saksela, E. et al., Immunological Rev., 71-123 (1979). Krishnaraj, R. Cell. Immuno l., 141, 306-322 (1992). NeutrophilsArnould, T. et al., Blood, 83, 3705-3716 (1994). Read, R.A. et al., Surgery, 114, 308-313 (1993). Conway, E.M. et al., Blood, 80,1254-1263 (1992). EosinophilsSchweizer, R.C. et al., Blood, 83, 3697-3704 (1994). Burgers, J.A. et al., Blood, 81, 49-55 (1993).Blom, M., et al., Blood, 83, 2978-2984 (1994).Kupffer CellsPage, D.T. and Garvey, J.S., J. Immunol. Methods, 27, 159-173 (1979).HepatocytesDou, M. et al., Cryobiol., 29, 454-469 (1992).Page, D.T. and Garvey, J.S., J. Immunol. Methods, 27, 159-173 (1979).Obrink, B. et al., Biochem. Biophys. Res. Comm., 77, 665-670 (1977).BasophilsKepley, C. et al., J. Immunol. Meth., 175, 1-9 (1994). Arock, M. et al., Int. Arch. Allergy Immunol., 102, 107-111 (1993).Tanimoto, Y. et al., Clin. Exper. Allergy, 22, 1020-1025 (1992).Leydig CellsSyed, V. et al., in vitro. J. Endocrinol. Invest., 14, 93-97 (1991).Schleicher, R.L. et al., Biol. Reprod., 48, 313-324 (1993).SpermatozoaChen. Y. et al., Int. J. Fertil., 37, 315-319 (1992). Bongso, A. et al., Archiv. Androl., 31, 223-230 (1993). Morales, P. et al., Human Reprod., 6, 401-404 (1991). Bone MarrowAvraham, H. et al., Blood, 79, 365-371 (1992). Genot, E. et al., Blood, 80, 2060-2065 (1992). Louache, F. et al., Blood, 78, 1697-1705 (1991). MacrophagesCalhoun, W.J., J. Lab. Clin. Med., 117, 443-452 (1991). Calhoun, W.J. et al., Am. Rev. Respir. Dis., 145, 317-325 (1992).Narahara, H., et al., Endocrinol. Metab, 77, 1258-1262 (1993).Mast CellsKulmburg, P.A. et al., Exp. Med., 176, 1773-1778 (1992).Inagaki, N. et al., Life Sci., 54, 1403-1409 (1994). Kurosawa, M. et al., Int. Arch. Allergy Immunol., 97, 226-228 (1992).ThymocytesHoshino. J. et al., Biochem. International, 27, 105-116 (1992).Fearnhead, H.O. et al., Biochem. Pharmacol., 48, 1073-1079 (1994).Sun, X.M. et al., Biochem. Pharmacol., 44, 2131-2137 (1992).Cohen, G.M. et al., J Immunol., 153, 507-516 (1994). Pancreatic IsletsBuitrago, A. et al., Biochem. Biophys. Res. Commun., 79, 823-828 (1977).EndothelialSbarbati, R. et al., Blood, 77, 764-769 (1991). NeuronsSoldenberg, S.S., and De Boni, U., J. Neurobiol., 14, 195-206 (1983).Platelet MembranesPerret, B. et al., Biochim. Biophys. Acta, 556, 434-446 (1979).Hepatocyte MembranesObrink, B. et al., Biochem. Biophys. Res. Commun., 77, 665-670 (1977).CHO MembranesCezanne, L. et al., Biochim. Biophys. Acta, 1112, 205-214 (1992).LysozomesLindley, E.R. and Pisoni, R.L., Biochem. J., 290, 457-462 (1993).Kominami, E. et al., J. Biochem., 111, 278-282 (1992). MitochondriaChemnitus, J.M. et al., Int. J. Biochem., 4, 589-596 (1993).Lopez-Mediavilla, C. et al., Exp. Cell Res., 203, 134-140 (1992).GranulesKjeldson, L. et al., Blood, 83, 1640-1649 (1994). Sengelov, H. et al., Biochem. J., 299,473-479 (1994). NucleiHahn, C. and Covault., J., Anal. Biochem., 190, 193-197 (1990).rbg 10/22/98Sigma brand products are sold through Sigma-Aldrich, Inc.Sigma-Aldrich, Inc. warrants that its products conform to the information contained in this and other Sigma-Aldrich publications. Purchaser must determine the suitability of the product(s) for their particular use. Additional terms and conditions may apply. Please see reverse side ofthe invoice or packing slip.。